Significance of T-lymphocytes in wound healing

To determine the importance of T-lymphocytes in wound healing, we examined the effect of T-lymphocyte depletion on the healing of surgical wounds. Thirty Balb/c mice were injected intraperitoneally with 1 mg of rat anti-mouse (IgG2b) cytotoxic monoclonal antibody (30H12) against the Thy1.2 (all T) determinant. Twenty-four hours later animals showed a greater than 95% depletion of Thy1.2 cells in peripheral blood and spleen. Thirty control mice received nonspecific rat immunoglobulin (1 mg). Twenty-four hours after treatment mice underwent a 2.5 cm dorsal skin incision with subcutaneous placement of polyvinyl alcohol sponges. Injections were repeated at weekly intervals. Wound healing was assessed at 2, 3, and 4 weeks by the breaking strength of wound strips and by the hydroxyproline content of sponge granulomas (an index of wound reparative collagen deposition). Thy1.2 depletion at death was 95% to 57% in peripheral blood and 86% to 68% in the spleen. Both groups gained weight equally. We found that T cell depletion significantly impairs wound breaking strength and wound collagen deposition at all times studied. The data strongly suggest that T-lymphocytes modulate fibroblast activity during normal wound healing.     

MHC-class-II-deficiency impairs wound healing

BACKGROUND: MHC-class-II-deficient mice lack T helper cell dependent immune reactions. T cell related immune functions are critical for normal wound healing. We hypothesized that MHC-II-deficiency compromises wound repair by affecting the normal wound immune response. MATERIAL AND METHODS: Groups of 10 male MHC-class II-knockout mice and wild-type controls underwent dorsal skin incision. Polyvinyl alcohol sponges were then inserted subcutaneously. The mice were sacrificed 10 days later to determine wound breaking strength and reparative collagen deposition. Activity of T cells and macrophages isolated from the spleens and from the healing wounds was investigated. Fibroblasts derived from the wounds were tested ex vivo for proliferative activity and collagen synthesis. RESULTS: Wound collagen deposition and wound breaking strength were impaired in MHC-class-II-knockout mice (P < 0.05). Impaired healing was reflected in diminished mitogen-reactivity of splenic T-cells (P < 0.01), and decreased CD4 expression in wounds. In addition, basal and LPS + IFN-gamma-induced synthesis of TNF-alpha and nitric oxide by wound-derived macrophages was impaired. Exvivo, fibroblast proliferation and fibroblast collagen production from MHC-II-deficient mice was decreased. CONCLUSION: MHC-II-deficiency compromises wound healing. This may be a reflection of impaired wound immune cell function and decreased activity of wound fibroblasts.     

Inhibition of wound repair by thymic hormones

To further define the role of the thymus in wound healing, we studied the effects of two thymic hormones on fibroplasia in normal euthymic and in nude athymic mice. Groups of 10 mice underwent a 2.5 cm dorsal skin incision with subcutaneous placement of polyvinyl alcohol sponges. Starting on the day of wounding, the following daily injections were given: (1) thymopentin (TP5), an active synthetic pentapeptide of thymopoietin, a naturally occurring thymic hormone (1 microgram/day/IM); (2) thymulin or facteur thymique serique (FTS), a naturally occurring circulating thymic hormone (0.2 microgram/day/IM); (3) control saline solution (0.1 ml/day/IM). All mice were killed 4 weeks after wounding, and wound breaking strength and hydroxyproline content of the sponge granulomas were measured. The results show that both thymic hormones impaired wound breaking strength and reparative collagen synthesis in normal and athymic mice. The magnitude of the wound healing impairment induced by the two hormones was equal in the thymus-bearing and in the nude mice. The data support previous findings, which suggested that the thymus has an inhibitory effect on wound healing.     

Supplemental dietary arginine enhances wound healing in normal but not inducible nitric oxide synthase knockout mice

BACKGROUND: Although generation of nitric oxide (NO) from inducible nitric oxide synthase (iNOS) has been shown to be required for cutaneous wound healing, no differences have been noted in incisional healing between iNOS knockout (iNOS-KO) and wild type (WT) mice. Because supplemental dietary arginine enhances cutaneous healing in normal rodents and is the sole substrate for NO synthesis, we studied whether arginine can enhance cutaneous wound healing in iNOS-KO mice. METHODS: Twenty iNOS-KO and 20 WT mice, all on a C57BL/6 background, were divided into 4 groups of 10 animals each. Ten animals with each trait were randomized to receive either normal food and tap water or food and water each supplemented with 0.5% arginine (w/w). All animals underwent a 2.5-cm dorsal skin incision with implantation of four 20-mg polyvinyl alcohol sponges into subcutaneous pockets. On postoperative day 14 the animals were killed. The dorsal wound was harvested for breaking strength determination and the wound sponges were assayed for hydroxyproline content and total wound fluid nitrite/nitrate concentration. RESULTS: Dietary arginine supplementation enhanced both wound breaking strength and collagen deposition in WT but not iNOS-KO mice. Wound fluid nitrite/nitrate levels were higher in WT than iNOS-KO animals but were not significantly influenced by additional arginine. CONCLUSIONS: These data demonstrate that supplemental dietary arginine enhances wound healing in normal mice. The loss of a functional iNOS gene abrogates the beneficial effect of arginine in wound healing. This suggests that the metabolism of arginine via the NO pathway is one mechanism by which arginine enhances wound healing.     

Interleukin 2 enhances wound healing in rats

Antigen-stimulated lymphocytes secrete lymphokines which have been shown to enhance in vitro fibroblast migration, proliferation, and protein synthesis. In the present experiments, the effect of human recombinant interleukin 2 (RIL-2) on wound healing was assessed in vivo. Groups of male Lewis rats, 225-250 g, underwent intraperitoneal insertion of osmotic pumps and a 7-cm dorsal skin incision with subcutaneous placement of polyvinyl alcohol sponges under anesthesia. The dorsal wounds were closed with stainless-steel sutures. The dose of RIL-2 administered was 60,000 u/rat/day for 7 days in experiment I, and 140,000 u/rat/day for 7 days in experiment II. Controls received equal volumes of excipient. Animals were sacrificed 10 days post wounding and wound healing was assessed by fresh breaking strength, fixed breaking strength (following 72 hr of Formalin fixation which maximally crosslinks the collagen present), and sponge hydroxyproline content (an index of reparative collagen accumulation). In vivo RIL-2 administration significantly augmented wound fresh and fixed breaking strength and wound collagen synthesis. Higher doses of RIL-2 (experiment II) did not result in further increases in the parameters studied. The data suggest that lymphocytes participate directly in the process of wound healing.     

L-arginine improves wound healing after trauma-hemorrhage by increasing collagen synthesis

BACKGROUND: Several studies indicate impaired wound healing after trauma and shock. Wound immune cell dysfunction seems to be responsible for altered wound healing after trauma-hemorrhage (T-H). In this respect, administration of the amino acid L-arginine normalized wound immune cell function under those conditions. It remains unknown, however, whether L-arginine improves impaired wound healing after T-H. METHODS: To study this, male C3H/HeN mice were subjected to a midline laparotomy (i.e., soft tissue trauma induced), and polyvinyl sponges were implanted subcutaneously at the wound site before hemorrhage (35 +/- 5 mm Hg for 90 minutes) or were subjected to sham operation. During resuscitation, mice received 300 mg/kg body weight L-arginine or saline (vehicle). Seven days thereafter, hydroxyproline (OHP), a metabolite of collagen synthesis, was measured in the wound fluid using high-performance liquid chromatography. Collagen types I and III were determined in the wound by Western blot analysis. In addition, wound breaking strength was measured 10 days after T-H or sham operation. RESULTS: The results indicate that OHP was significantly decreased in T-H mice. L-arginine, however, restored depressed OHP in the wound fluid in the T-H animals. Similarly, L-arginine treatment prevented a significant depression of collagen I synthesis after T-H. Collagen III was not significantly affected by T-H or L-arginine. Most important, L-arginine increased maximal wound breaking strength after severe blood loss. Therefore, L-arginine improves wound healing after T-H by increasing collagen synthesis. CONCLUSION: Because L-arginine improves wound healing, the results suggest that L-arginine might represent a novel and useful adjunct to fluid resuscitation for decreasing wound complications after trauma and severe blood loss.     

Nitric oxide enhances experimental wound healing in diabetes

BACKGROUND: Diabetes is characterized by a nitric oxide deficiency at the wound site. This study investigated whether exogenous nitric oxide supplementation with the nitric oxide donor molsidomine (N-ethoxycarbomyl-3-morpholinyl-sidnonimine) could reverse the impaired healing in diabetes. METHODS: Wound healing was studied by creating a dorsal skin incision with subcutaneous polyvinyl alcohol sponge implantation in diabetic and non-diabetic rats. Half of each group was treated with molsidomine. Collagen metabolism was assessed by wound breaking strength, hydroxyproline (OHP) content, RNA expression for collagen type I and III, and matrix metalloproteinase (MMP) 2 activity in wound sponges. Wound fluid, plasma and urinary nitric oxide metabolite levels, and the number of inflammatory cells were assessed. RESULTS: OHP content and wound breaking strength were significantly increased by molsidomine. MMP-2 activity in wound fluid was decreased in diabetes and upregulated by nitric oxide donors. The impaired inflammatory reaction in diabetes was unaffected by nitric oxide donor treatment and ex vivo nitric oxide synthesis was no different between wound macrophages from control and diabetic animals, suggesting that the nitric oxide deficiency in the wound is due to a smaller inflammatory reaction in diabetes. CONCLUSION: The nitric oxide donor molsidomine can at least partially reverse impaired healing associated with diabetes.     

Regulation of nitric oxide synthesis in wounds by IFN-gamma depends on TNF-alpha.

Macrophage-derived nitric oxide is a critical mediator in wound healing. Its regulation in vivo, however, remains unclear. We hypothesized that interferon (IFN)-gamma plays an important role in the regulation of nitric oxide in wounds. Groups of 12 male IFN-gamma -knockout mice and wild-type controls underwent dorsal skin incision and polyvinyl alcohol sponges were inserted subcutaneously. Mice were sacrificed 10 days later to determine wound breaking strength and reparative collagen deposition. Synthesis of nitric oxide (NO), tumor necrosis factor (TNF)-alpha, and IFN-gamma was measured in the wound. Wound-derived macrophages were tested for NO synthesis in the presence or absence of IFN-gamma, TNF-alpha, and anti-TNF-alpha antibody. In a separate experiment, IFN-gamma -knockout mice and wild-type controls were treated with molsidomine, a nitric oxide donor. It was found that wound collagen deposition and wound breaking strength were impaired in IFN-gamma-knockout mice (p < .05). Impaired healing was reflected in diminished synthesis of TNF-alpha and NO in wounds (p < .05). In vivo treatment with molsidomine reversed impaired healing in IFN-gamma-deficient mice. Ex vivo, addition of IFN-gamma stimulated the synthesis of TNF-alpha and NO in wound-derived macrophages. IFN-gamma -induced NO synthesis by wound-derived macrophages was abolished by anti-TNF-alpha-antibody-treatment, which could be fully reversed by exogenous TNF-alpha. Thus we conclude that IFN-gamma-deficiency impairs wound healing and diminishes NO synthesis in wound-derived macrophages. The stimulatory effect of IFN-gamma on macrophage NO production depends on endogenous TNF-alpha synthesis.     

The effect of in vivo T helper and T suppressor lymphocyte depletion on wound healing.

The role of T lymphocytes in wound healing is still not well-defined. Because it had been previously shown that in vivo depletion of T cells leads to impaired wound healing, the effect of depleting T cell subsets on subsequent fibroplasia was studied. T helper/effector cells were depleted by the use of the monoclonal antibody GK1.5, reactive against the L3T4 antigen (CD4). T suppressor/cytotoxic lymphocytes were depleted by using the 2.43 monoclonal antibody reactive against the Lyt 2 antigen (CD8). In the first experiment, Balb/c mice were treated with the antibodies starting at 24 hours before wounding was performed, and weekly thereafter. Depletion of the T helper/effector cells had no effect on wound-breaking strength or hydroxyproline deposition in sponge granulomas, whereas depletion of T suppressor/cytotoxic cells significantly enhanced both of these healing parameters. In a second experiment, T cell subset depletion was started on Days 0, 3, 7, 10, and 14 postwounding, and treatments were continued weekly thereafter. Once again, depletion of T helper/effector cells had no effect on wound healing, whereas depletion of T suppressor/cytotoxic cells markedly increased both wound-breaking strength and collagen synthesis. In conclusion, the data show that T suppressor/cytotoxic cells have a counter-regulatory role in wound healing, whereas the T cell subset responsible for up-regulating wound healing remains to be identified.     

Effect of supplemental ornithine on wound healing

BACKGROUND: Supplemental arginine has been shown to enhance wound healing, in particular collagen synthesis. Ornithine is the main metabolite of arginine in the urea cycle and shares many of the biopharmacologic effects of arginine. The present study examines the effect of ornithine supplementation on wound healing and attempts to describe its possible mechanism. METHODS: Wild type (WT) and iNOS knockout (KO) mice were randomized to receive either normal chow and tap water or chow and water each supplemented with 0.5% ornithine (w/w). All animals underwent a midline dorsal skin incision with implantation of polyvinyl alcohol sponges into subcutaneous pockets. On postoperative day 14 the animals were sacrificed. The dorsal wound was harvested for breaking strength determination while the wound sponges were assayed for hydroxyproline content, total wound fluid amino acid, and nitrite/nitrate (NOx) concentration. RESULTS: Dietary ornithine supplementation enhanced wound breaking strength and collagen deposition in both WT and KO mice. This was accompanied by increased wound fluid proline and ornithine levels but not arginine, citrulline, or NOx levels. CONCLUSIONS: The results from this study demonstrate that ornithine supplementation enhances wound healing in both WT and KO mice. This suggests that ornithine's effect on wound healing is independent of the iNOS pathway.     

Regulation of Nitric Oxide Synthesis in Wounds by IFN-γ Depends on TNF-α

Macrophage-derived nitric oxide is a critical mediator in wound healing. Its regulation in vivo, however, remains unclear. We hypothesized that interferon (IFN)-γ plays an important role in the regulation of nitric oxide in wounds. Groups of 12 male IFN-γ -knockout mice and wild-type controls underwent dorsal skin incision and polyvinyl alcohol sponges were inserted subcutaneously. Mice were sacrificed 10 days later to determine wound breaking strength and reparative collagen deposition. Synthesis of nitric oxide (NO), tumor necrosis factor (TNF)-α, and IFN-γ was measured in the wound. Wound-derived macrophages were tested for NO synthesis in the presence or absence of IFN-γ, TNF-α, and anti-TNF-α antibody. In a separate experiment, IFN-γ -knockout mice and wild-type controls were treated with molsidomine, a nitric oxide donor. It was found that wound collagen deposition and wound breaking strength were impaired in IFN-γ-knockout mice (p <. 05). Impaired healing was reflected in diminished synthesis of TNF-α and NO in wounds (p <. 05). In vivo treatment with molsidomine reversed impaired healing in IFN-γ-deficient mice. Ex vivo, addition of IFN-γ stimulated the synthesis of TNF-α and NO in wound-derived macrophages. IFN-γ -induced NO synthesis by wound-derived macrophages was abolished by anti-TNF-α-antibody-treatment, which could be fully reversed by exogenous TNF-α. Thus we conclude that IFN-γ-deficiency impairs wound healing and diminishes NO synthesis in wound-derived macrophages. The stimulatory effect of IFN-γ on macrophage NO production depends on endogenous TNF-α synthesis.     

Supplemental L-arginine enhances wound healing in diabetic rats

L-arginine has been shown to enhance wound strength and collagen deposition in rodents and humans. Diabetes mellitus, which impairs wound healing, is accompanied by a reduction in nitric oxide at the wound site. The amino acid L-arginine is the only substrate for nitric oxide synthesis. We sought to determine whether supplemental L-arginine can restore the impaired wound healing of diabetic rats. Fifty-six male Lewis rats were used in this study, of which twenty-nine rats were rendered diabetic 7 days prior to surgery with intraperitoneal streptozotocin. Twenty-seven untreated rats served as controls. Animals underwent a dorsal skin incision with implantation of polyvinyl-alcohol sponges. Sixteen diabetic and 14 normal rats received 1 g/kg/day of L-arginine by injection, while the remainder received saline injections only. Animals were euthanized 10 days postwounding, and their wounds were analyzed for breaking strength. The wound sponges were assayed for total hydroxyproline and nitrite/nitrate content. Plasma and wound fluid concentrations of L-arginine, ornithine, and citrulline were determined. Wound sponge RNA was extracted and subjected to Northern blot analysis for procollagen I and III. Diabetic wounds had greatly decreased breaking strengths compared with controls. L-arginine significantly enhanced wound breaking strengths in both control (+23%) and diabetic animals (+44%), and also increased wound hydroxyproline levels in both diabetic (+40%) and control animals (+24%) as compared to their saline-treated counterparts. mRNA for procollagen I and III were elevated by L-arginine treatment in both diabetic rats and controls. Treatment with L-arginine significantly increased wound fluid nitrite/nitrate levels in diabetic animals. The data show that the impaired healing of diabetic wounds can be partially corrected by L-arginine supplementation, and that this effect is accompanied by enhanced wound nitric oxide synthesis.     

The effect of testosterone propionate on wound healing in normal and castrate rats

The dependence of wound healing on testosterone was studied in normal and castrate rats by determination of wound breaking strength (WBS) in dermal wounds, by implantation of subcutaneous polyvinyl sponges (PVS) and by [³H]proline tracer studies. The level of testosterone achieved with various doses of testosterone propionate (TP) was assessed using the androgenic effect of this hormone on prostate and seminal vesicle weights. Exogenous testosterone propionate (0.25 – 3.0 mg/day) produced no acceleration of wound healing as measured by WBS on 14- and 21-day wounds. In castrate rats a mild inhibition of healing (15% decrease in WBS) was found in 14-day wounds but no difference was found between castrate and control in 21-day wounds. The rate of wound collagen synthesis was assessed by measuring the conversion of [³H]proline to [³H]hydroxyproline, a process essentially limited to procollagen synthesis. It was not altered by castration, or by administration of testosterone propionate (0.0625 – 1.0 mg/day) to castrate rats. Similarly, deposition of tissue in polyvinyl sponges whether measured as added dry weight or total hydroxyproline did not differ significantly between control and castrate rats receiving testosterone propionate (0–1.0 mg/day). As a method of assessing wound healing, WBS measurements produced the most consistent results. In conclusion, no longterm dependence of wound healing on testosterone was identified in the testosterone-depleted (castrate) rat although some early depression was noted, and no acceleration of the normal process resulted from exogenous testosterone administration in the normal or testosterone-depleted rat.     

Intravenous hyperalimentation with high arginine levels improves wound healing and immune function

The purpose of this study was to evaluate the effect of increased arginine levels in intravenous hyperalimentation (IVH) therapy on wound healing and thymic immune function. Groups of SD rats, 275-325 g, underwent placement of internal jugular catheter, 7-cm dorsal skin wounding, insertion of polyvinyl alcohol sponges subcutaneously, and closure of wounds with stainless-steel sutures. Twenty-four hours later, rats were started on IVH at a rate of 0.8-1 ml/100 g body wt/hr. All IVH solutions contained 20% dextrose, adequate amounts of minerals and vitamins, and two different amino acid mixtures: (A) Fre III (4.05 g ARG/liter) (n = 13); (B) experimental (7.50 g ARG/liter) (n = 11). Solutions were isonitrogenous, and contained similar amounts of essential amino acids. After 7 days of IVH, weight gain did not differ between the two groups; however, cumulative N balance was superior in group A. Wound healing was improved in group B as assessed by fresh wound strip breaking strength, fixed breaking strength, and the amount of reparative collagen deposition as assessed by the hydroxyproline content of the implanted sponges. Group B animals also had improved thymic function as assessed by thymic weight, the total number of thymic lymphocytes/gland and mitogenic reactivity of thymic lymphocytes to PHA and Con A. The experiments indicate that high arginine levels in IVH solutions improve wound healing and thymic immune function following injury.     

Thymic inhibition of wound healing: Abrogation by adult thymectomy

It has been previously observed that the thymus and wound respond in a similar manner, i.e., agents that enhance thymic function increase wound healing, while factors which decrease thymic function impair healing. In order to elucidate if the thymus has a direct influence on wounds, we have studied wound healing in adult rats who have undergone thymectomy at 4–8 weeks of age. In three separate experiments we found that thymectomized rats had fresh wound breaking strengths significantly greater than sham-thymectomized rats. There were no differences noted in the amount of reparative collagen accumulated in subcutaneously implanted polyvinyl alcohol sponges or in the breaking strength of wound strips fixed in 10% formalin, which maximally cross-links the collagen present; the ratios of fixed to fresh wound breaking strengths were significantly greater in sham-thymectomized rats. Rats who had undergone thymectomy with immediate intraperitoneal placement of Millipore chambers containing autologous thymic fragments had wound breaking strengths similar to sham-thymectomized or intact animals. We conclude that thymectomy at 4–8 weeks of age increases wound maturation and collagen cross-linking. This suggests that the thymus normally has an inhibitory effect on wound healing and a role of T-suppressor cells on this process is postulated.     

The Impairment of Wound Healing Process is Correlated With Abnormalities of TNF-α Production by Peritoneal Exudate Cells in Obstructive Jaundiced Rats

The wound healing process and production of tumour necrosis factor alpha (TNF-α) by peritoneal cells of 7-day and 14-day obstructive jaundice (OJ) and sham-operated rats were investigated. In the study the skin wound breaking strength was measured, In addition such histological and biochemical parameters as fibroblast and endothelial cell proliferation, inflammatory cell infiltration and hydroxyproline content were evaluated in polyurethane sponge discs implanted subcutaneously into rats. TNF-α production by peritoneal exudate cells (PEC), both spontaneous and lipopolysaccharide (LPS)- induced was determined by a bioassay. In OJ rats the process of both early as well as late phase of healing was impaired. The breaking strength of skin wound was decreased, the fibroblast and endothelial cell proliferation and collagen deposition, as well as hydroxyproline content were diminished. In 7 day OJ the numbers of inflammatory cells in the implants were lowered with a subsequent slight increase on day 14 of OJ. The spontaneous and LPS induced TNF- α production by PEC were significantly higher in 7 day OJ as compared with sham-operated controls. On day 14 of OJ the LPS-induced TNF-α level was, in contrast, much lower and did not differ much from the spontaneous TNF-α production. We conclude that the impairment of wound healing in OJ results from disturbances in functioning of the immune system caused by systemic endotoxaemia.     

Supplemental vitamin A prevents the tumor-induced defect in wound healing.

To test our hypothesis that supplemental vitamin A would mitigate the impaired healing that occurs in tumor-bearing animals, six groups of C3H mice, eight per group, eating a standard commercial mouse chow ad libitum that supports normal growth, reproduction, and longevity were innoculated with 200,000 C3HBA cells. When tumors measured approximately 6 mm in diameter, the mice were anesthesized and wounded (dorsal skin incisions and subcutaneous polyvinyl alcohol sponges). Twenty-four hours later, two groups (one continued on the chow and the other started on the chow supplemented with 150,000 IU vitamin A/kg chow) underwent local tumor irradiation; two groups, one ingesting the chow, the other the vitamin A supplemented chow, were started on cyclophosphamide therapy; two groups, one ingesting the chow, the other the vitamin A supplemented chow, received neither local tumor irradiation nor cyclophosphamide therapy. An additional two groups ingesting the chow, one group neither innoculated with tumor nor wounded, the other wounded by not innoculated, served as controls. Wound breaking strength and sponge reparative collagen accumulation (assessed by hydroxyproline proline measurement) were used as indicators of wound healing. The mice were killed 12 days after wounding. Tumor presence decreased wound breaking strength and sponge hydroxyproline content; these effects were largely negated by supplemental vitamin A. Local tumor irradiation diminished the adverse effect of tumor on sponge reparative collagen content but to a lesser extent than the supplemental vitamin A. Supplemental vitamin A added to the irradiation effect on healing but irradiation did not add to the vitamin A effect. Cyclophosphamide, a systemic radiomimetic anti-tumor agent, did not alter the impaired wound healing of the tumor-bearing mice. Supplemental vitamin A mitigated the impaired wound healing in the cyclophosphamide-treated tumor-bearing mice. Supplemental vitamin A also moderated the effects of wounding, tumor, and tumor therapies (local irradiation and cyclophosphamide) on the increase in adrenal size, leukopenia, thrombocytopenia, and thymic involution (except the last was not moderated in the cyclophosphamide-treated tumor-bearing rats). The splenic enlargement in the untreated tumor-bearing wounded rats and in those treated with cyclophosphamide was lessened by supplemental vitamin A. We hypothesize that these anti-stress effects of vitamin A underlie, in part, its action in mitigating the impaired wound healing of tumor-bearing mice, including those treated by local irradiation or cyclophosphamide. These findings have implications for the care of patients with malignant tumors.     

Wound healing and T-lymphocytes

T-cell depletion leads to impaired wound healing. We studied the effect of combined T-helper and T-suppressor lymphocyte depletion on wound healing and compared it with the effect of all T-cell depletion. Groups of 10 male balb/c mice, 8 weeks old, underwent a 2.5-cm skin incision and subcutaneous implantation of polyvinyl alcohol sponges. Twenty-four hours prior to wounding one group was treated with 3OH12, a rat anti-mouse monoclonal antibody against the Thy-1.2 antigen present on all T-cells (1 mg); another group received 1 mg each of GK1.5 (anti-L3T4, CD4; anti-helper/effector subset) and 2.43 (anti-Lyt 2.1, CD8; anti-suppressor/cytotoxic subset). All monoclonal antibodies are cytotoxic in vivo. Controls received 1 mg of nonspecific rat IgG. Treatments were repeated weekly. Animals were sacrificed at 2 and 4 weeks postwounding. Equal depletion of all T- and Th- and Ts-subsets in peripheral blood and spleens was noted in the two experimental groups at sacrifice. Depleting Thy-1.2 cells (all T-cells) impaired wound healing as assessed by wound breaking strength and collagen synthesis. Combined anti-T-helper/effector and T-suppressor/cytotoxic depletion resulted in improved wound-healing parameters. This suggests that there is a Thy-1.2+, L3T4-, Lyt2- subpopulation of T lymphocytes which normally stimulates wound healing.     

Inhibition of tumor necrosis factor-alpha attenuates wound breaking strength in rats.

Exogenous administration of tumor necrosis factor-alpha has been shown to both enhance and attenuate cutaneous healing in a dose-dependent manner. We examined the effects of tumor necrosis factor inhibition in the healing wound by both systemic and local administration of tumor necrosis factor-binding protein. Male Balb/C mice underwent dorsal skin incision with subcutaneous implantation of 20 mg polyvinyl alcohol sponges (4 per animal). In Experiment I, one group (n = 20) received intraperitoneal injections of tumor necrosis factor-binding protein (3 mg/kg) at the time of wounding, while another group (n = 20) received saline. Four animals from each group were euthanized on days 1, 3, 5, 7, and 14 postwounding. In Experiment II, one group (n = 10) received an intraperitoneal injection of tumor necrosis factor-binding protein (3 mg/kg) at the time of wounding and every third day thereafter. Another group (n = 10) received an intraperitoneal injection of saline at the time of wounding and every third day thereafter. In Experiment III, one group received a single intraperitoneal injection of tumor necrosis factor-binding protein (3 mg/kg) at the time of wounding (n = 7), or on postwounding day 4 (n = 7), or day 7 (n = 7). Another group received saline injections at the time of wounding (n = 7), or on postwounding days 4 or 7 (n = 7, respectively). All animals in Experiments II and III were killed at postwounding day 14. Wound breaking strengths were assessed using a tensiometer. Wound fluid collected from the implanted sponges was assayed for tumor necrosis factor-alpha and tumor necrosis factor-binding protein levels using a biological assay and enzyme-linked immunosorbent assay, respectively. Collagen gene expression in sponge granulomata was assessed by Northern analysis. Collagen deposition in sponges was quantified by measuring hydroxyproline content. Wounds were significantly weaker in the animals that received repeated injections of tumor necrosis factor-binding protein with a mean wound breaking strength of 93.1 g vs. 186.6 g in controls (p < 0.05). Wound breaking strength in groups that received a single injection of tumor necrosis factor-binding protein on either day 0, 4, or 7 postwounding were no different than their respective controls. There was no difference in the mean hydroxyproline content of sponges between any of the tumor necrosis factor-binding protein groups and their respective controls. Northern analysis for collagen I and III expression also revealed no differences. These data indicate that continued systemic administration of tumor necrosis factor-binding protein resulted in significantly weaker wounds with no corresponding differences in wound collagen content, and collagen gene expression. This suggests that tumor necrosis factor-alpha inhibition throughout healing leads to a qualitatively impaired wound without a quantitative alteration in collagen deposition.     

Colchicine and wound healing

Rats with incised and sutured wounds and having subcutaneously implanted polyvinyl alcohol sponges were treated with various doses of colchicine. Breaking strength of wound tissue and biochemical analysis of sponge-induced granuloma tissue were compared with similar measurements in pair-fed controls. Colchicine reduced breaking strength of wound scar tissue significantly while lysyl oxidase activity in sponge tissue was not affected. Synthesis of collagenous and noncollagenous protein was stimulated by colchicine but accelerated collagen synthesis did not result in increased deposition of collagen in sponge tissue. The increased urinary excretion of hydroxyproline in colchicine-treated rats was paralleled by loss of body weight. Colchicine-induced cytotoxicity was also identified in fibroblasts from sponge granuloma tissue examined by transmission electron microscopy. We conclude that some reduction of total collagen deposition and breaking strength of wound tissue in colchicine-treated animals may have been the result of general toxicity of the drug. A specific effect of colchicine on collagen metabolism cannot be ruled out.